Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without β-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or β-estradiol (20 ng/ml) or relaxin plus β-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and β-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants – a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or β-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and β-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and provide evidence for a link between relaxin, MMPs, and matrix degradation. In certain sites in and around joints, ligaments and tendons subjected to complex tensile and compressive loading specialize into fibrocartilaginous tissues -3 contaiTo understand the potential basis for relaxin and estrogen's modulation of the composition of fibrocartilaginous tissues, we previously studied cells isolated from rabbit TMJ discs. Relaxin induced the expression of the matrix metalloproteinases (MMPs) collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) in a dose-dependent fashion but had little effect on the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) or TIMP-2 . In cellOne such disease is TMJ disorders, which affect some 11 million adults in the USA ,14, predIn this study we determined the effects of relaxin with or without β-estradiol on the modulation of MMPs, and assessed the contribution of these proteinases to the changes in collagen and glycosaminoglycan (GAG) content in fibrocartilaginous disc explants. Our findings are consistent with the hypothesis that relaxin-mediated induction of MMPs is associated with the loss of matrix macromolecules that could compromise tissue function and biomechanics and might lead to joint disease.35S was from Amersham . Protein assay kits, gelatin (EIA grade), and nitrocellulose membrane were from Bio-Rad . α-Minimal essential medium, trypsin, penicillin–streptomycin, and Fungizone® were from Gibco . All other standard chemicals were from Sigma or Fisher Scientific .Twenty-week-old female New Zealand white rabbits were obtained from Nita Bell Laboratories . Ketamine hydrochloride was from Parke Davis , and xylazine was from Rugby Lab . Lactalbumin hydrolysate, α-casein, β-estradiol-17-valerate, pepsin, papain, chondroitin sulfate A sodium from bovine trachea, Safranin-O, Fast Green, cetylpyridinium chloride, and other reagents were from Sigma . 1,9-Dimethylmethylene blue (DMMB) was from Molecular Probes , and Rabbit anti-human collagenase-1 polyclonal antibody and rabbit anti-mouse stromelysin-1 monoclonal antibody, horseradish peroxidase-conjugated secondary antibodies, and the MMP inhibitor GM6001 and its control analog were from Chemicon International . Rabbit anti-human-TIMP-1 antibody that cross-reacts with the rabbit inhibitor  was from2.All procedures on rabbits were approved by the Committee on Animal Research of the University of California, San Francisco, and conducted in accord with accepted standards of humane animal care. Rabbits were anesthetized with ketamine hydrochloride (40 mg/kg) and xylazine (3–5 mg/kg), and the TMJ discs were harvested bilaterally under sterile conditions and immediately placed in calcium-free and magnesium-free phosphate-buffered saline (PBS) containing antibiotics . After removal of the synovium under a dissecting microscope, each disc was washed three times in PBS and bisected longitudinally such that four samples from each rabbit were available (three for hormone treatments and one for control). The hemisections were weighed, placed in wells of a 24-well culture plate, covered with 1 ml of serum-free medium  with or without hormones, and cultured at 37°C in air containing 5% CO®, weighed, digested, and used for the determination of GAG and collagen content.For determination of MMPs and GAG staining, 32 hemisections from eight rabbits were exposed to medium alone, β-estradiol (20 ng/ml), relaxin (0.1 ng/ml), or both hormones at the same doses for 48 hours. The conditioned medium was collected and stored for MMP assays, and the discs were processed for GAG staining. To assess the contribution of relaxin-induced MMPs to the loss of collagen and GAGs, 24 hemisections from six rabbits were cultured with the MMP inhibitor GM6001 or its control analog 2 hours before and during the hormone treatments. The inhibitor was used at 10 μM, because this concentration was shown to inhibit collagenase activity induced by 0.1 ng/ml relaxin in dose–response experiments to baseline levels. The conditioned medium was collected and stored at -70°C for total protein and MMP assays. The discs were dried in a SpeedVacTo determine whether the observed induction of collagenase by relaxin is specific to fibrocartilage, experiments were performed with pubic symphysis fibrocartilage, which is a known target site for β-estradiol and relaxin as a positive control, and with articular cartilage from the knee. For retrieval of articular cartilage, the joint was shaved, the articular surfaces were exposed, and the cartilage was scraped from the articular surfaces of the femur and tibia and incubated in PBS with antibiotic as described above. Similarly, the pubic bones and symphyseal areas were exposed under sterile conditions and the pubic symphysis (fibrocartilaginous tissues between the pubic bones) was dissected, removed, and incubated in PBS with antibiotics. The tissues were weighed, placed in wells of a 24-well culture plate, and studied as described above.Hormone-induced changes in collagenase-1, stromelysin-1, and TIMP-1 were determined by western blotting. Disc-conditioned medium was mixed with 4 × sample buffer and subjected to SDS–polyacrylamide-gel electrophoresis with 10% or 18% gels. Equal amounts of protein (determined with a bicinchoninic acid protein assay kit) were loaded in each lane. The proteins were transferred to nitrocellulose membranes, which were blocked, washed, and incubated for 1 hour with antibodies against TIMP-1 (1:250 dilution), collagenase-1 , or stromelysin-1 (1:500 dilution). The membranes were then washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1000 dilution), and washed again. Bands were revealed by incubation with enhanced chemiluminescence reagent and exposure to radiographic film. The bands for TIMP-1 western blots were quantified by videodensitometry as described . Conditi2, 0.02% NaN3), stained with 5% Coomassie blue, and destained in 10% acetic acid and 40% methanol until proteinase bands were clearly visible. Images of the gels were captured with a charge-coupled device camera and NIH image software. The levels of 53/58 kDa gelatinolytic and 51/54 kDa caseinolytic enzymes and their low-molecular-mass activated forms were quantified by videodensitometry [Enzyme activities were quantified by substrate zymography of conditioned media from 32 hemisections (mean wet weight 13 ± 9 mg). The samples were standardized by total protein and subjected to SDS–polyacrylamide-gel electrophoresis with 10% gels containing 2 mg/ml gelatin or casein at 15°C as described . The gelitometry . The subitometry -25. In aitometry .To assess changes in GAG levels, the discs were washed three times in PBS, frozen in OCT compound, and sectioned with a cryostat. The section were defrosted for 30 min, fixed for 10 min in methanol, air-dried for 15 min, stained with 1% Fast Green solution for 3 min, placed in 1% acetic acid for 1 min, stained with 2% Safranin-O for 2 min, dehydrated through successive ethanol and xylene washes, and mounted with coverslips. Ten sections of each hemisection were analyzed by an examiner blinded to the hormone treatment. The stained discs were videodigitized and analyzed with a software program that automatically outlined the total and Safranin-O-stained areas with threshold settings . These areas were then quantified with NIH Image 1.62, and the percentage of disc staining positive for GAGs was calculated from the ratio of the stained area to the total area in each section. The average of the 10 values for each half disc was used for analysis.35S as described [g for 20 min), the supernatant was removed and the precipitate was dissolved in 600 μl of concentrated formic acid by heating to 70°C for 10 min. Aliquots (20 μl) of this solution were added to 3 ml of scintillation fluid and subjected to liquid scintillation counting. The radioactivity (counts/min) was standardized to the total dry disc weight.To quantify GAG biosynthesis, 32 disc hemisections (mean weight 14 ± 4 mg) were incubated at 37°C for 6 hours in 1 ml of phenol-free and serum-free medium with or without hormones and 165 kBq (0.0044 mCi) of escribed . The disEach disc hemisection was digested in 600 μl of 3 mg/ml pepsin in 0.05 M acetic acid and incubated at 37°C for 18–20 hours in a dry bath. DMMB binding assays for GAGs, and Sircol assays for collagen content, were performed in triplicate on 24 disc hemisections. The DMMB reagent was prepared as described . Pepsin g to separate the unbound dye from the collagen-bound dye. After removal of the unbound dye, 1 ml of the alkali reagent was added to the collagen–dye complex and vortex-mixed to dissolve the collagen-bound dye completely. Aliquots (200 μl) were transferred to the 96-well plates, and absorbance at 550 nm was determined with a microtiter plate reader . The collagen concentration (μg/ml) was determined against a collagen standard curve, and the disc collagen content was standardized to the total disc dry weight.For the collagen assay, 200 μl of pepsin digest was mixed with 1 ml of Sircol dye reagent, incubated for 30 min at room temperature, and centrifuged at 10,000 Collagenase activity in conditioned medium from discs cultured with GM6001 or control analog was assessed by FITC–collagen assay. A 96-well plate was coated with FITC–collagen (10 μg per well) overnight at 4°C and washed twice with PBS. Disc-conditioned medium (100 μl) was added to the wells, and the plate was incubated at 35°C for 1 hour. As a reference, 100 μl of blank medium containing 3000 ng of bacterial collagenase was added to one set of wells for complete digestion of FITC–collagen. After incubation, 90 μl from each well was transferred to another 96-well plate, and the fluorescence intensity of degraded FITC–collagen products was determined with a microplate spectrofluorometer  with excitation at 494 nm and emission at 518 nm. The data were converted to relative fluorescence units of collagenase activity as described by the manufacturer and standardized to the dry weight of each half disc. The fold differences in collagenase activity in medium from control and hormone-treated discs were determined for each experiment. All assays were performed in duplicate.P < 0.05 was considered statistically significant. Values are expressed as means ± SD.Because of inherent variability in matrix content and proteinase activity in discs from different rabbits, three disc hemisections from each rabbit were treated with hormones and one served as control. MMP levels and the GAG and collagen content in each hormone-treated disc hemisection were standardized to the values of the control hemisection within each animal and the fold changes were plotted as histograms. The statistical significance of differences was determined by single-factorial analysis of variance (ANOVA). Intergroup differences were analyzed by Fisher's multiple comparisons test; P < 0.05) and was not potentiated by β-estradiol  Fig. , lane 1,iol Fig. .P < 0.05) and was not potentiated by β-estradiol.Because the expression of stromelysin and collagenase is often coordinately regulated , we asseIn pubic symphysis fibrocartilage, which is a known target site for β-estradiol and relaxin, β-estradiol caused slight increases in collagenase-1, while relaxin alone or in combination with β-estradiol induced a substantially greater expression of collagenase-1 relative to untreated discs Fig. . RelaxinP < 0.01, ANOVA) in GAG staining between control discs and those treated with relaxin  or β-estradiol plus relaxin (P < 0.01). Similarly, the GAG-positive area was significantly smaller  in discs treated with relaxin  or β-estradiol plus relaxin (P < 0.05) than in those treated with β-estradiol alone.Because all hormone treatments induced stromelysin-1 expression in explanted discs, we assessed the level of a known substrate, proteoglycans, in Safranin-O-stained sections. The GAG-positive area was larger in control discs  and discs treated with β-estradiol (29.7 ± 4.7%) than in those treated with relaxin (19.2 ± 3.3%) or β-estradiol plus relaxin (16.9 ± 2.7%) Fig. . These fP < 0.05), differences between the other groups were not significant  twofold induction in TIMP-1 expression over controls . However, zymography showed increased 51/54 kDa caseinolytic activity (stromelysin-1) only in hormone-treated explants incubated with the control analog Fig. , and notP < 0.0001, ANOVA), which was 30% lower in relaxin-treated explants  and 40% lower in those treated with β-estradiol plus relaxin (P < 0.001) than in untreated explants  in discs treated with relaxin  or β-estradiol plus relaxin (P < 0.001) than in those treated with β-estradiol alone. In the presence of GM6001, however, hormone treatments did not affect the GAG content  only in the presence of the control analog and only by relaxin  or β-estradiol plus relaxin   Fig. . In the 01) Fig. .This study shows that relaxin induced the expression of collagenase-1 and stromelysin-1 in rabbit TMJ disc explants, accompanied by a loss of GAGs and collagen, but did not affect GAG synthesis. In explants cultured with the MMP inhibitor GM6001, collagenase-1 and stromelysin-1 activities in hormone-treated discs were inhibited to baseline levels, and collagen and GAG content were maintained at control levels. These findings show that relaxin has degradative effects on nonreproductive synovial joint fibrocartilaginous tissue and provide evidence that increases in MMP activity mediated by relaxin and β-estradiol plus relaxin contribute directly to the loss of disc collagen and GAGs. The lack of effect on GAG synthesis further validates the importance of the degradative component of the remodeling cycle in relaxin's modulation of matrix loss in fibrocartilage.Because the MMP inhibitor used in our studies is not specific for collagenase-1 and stromelysin-1, the hormone-induced loss of collagen and GAGs cannot be specifically linked to those two proteinases. Rather, our findings implicate MMPs in general in this response. However, because GM6001 has a low dissociation constant for both collagenase-1 and stromelysin-1 , and the35S incorporation, but it produced a statistically significant increase in TIMP-1 expression that could have counteracted any increases in degradative activity due to increased expression of collagenase-1 and stromelysin-1. Indeed, the results of the collagen degradation assay lend credence to this hypothesis. These findings imply that relaxin and β-estradiol selectively contribute to the degeneration of fibrocartilaginous tissue by differentially modulating MMP expression, matrix synthesis, and net matrix content.In contrast to the results obtained with relaxin and β-estradiol plus relaxin, the induction of collagenase-1 and stromelysin-1 by β-estradiol alone was not accompanied by changes in GAG or collagen content within the disc. How can we explain this apparent discrepancy? β-Estradiol had little effect on GAG synthesis, as measured by The potential similarities in the responsiveness of TMJ fibrocartilaginous explants and the pubic symphysis fibrocartilage to relaxin are reflected not only by the relaxin's induction of collagenase but also by the comparable loss of collagen on the exposure of these tissues to the hormone ,9. Thus,The response of articular cartilage to relaxin or β-estradiol plus relaxin was substantially different from that of the TMJ disc and pubic symphysis fibrocartilages. Although the reasons for these differences remain to be determined, it is well accepted that articular cartilage is a cartilaginous tissue containing chondrocytic cells, whereas fibrocartilage is a heterogenous tissue composed of cartilage and fibrous tissue that contains cells of fibroblastic, chondrocytic, and fibrochondocytic phenotypes. It is plausible that of these cells, the fibroblastic and/or fibrochondrocytic cells found in fibrocartilage, rather than the chondrocytic cells, are those that produce the observed responses to relaxin and β-estradiol plus relaxin. Indeed, previous findings on both dermal fibroblasts showing a potent induction of MMP-1  and on aOur findings are consistent with emerging data suggesting that the mechanisms for the loss of matrix macromolecules caused by relaxin are tissue-specific . Thus, fin vivo showing relaxin-mediated decreases in GAG concentration in the TMJ discs of ovariectomized rabbits suggest that this systemic hormone can indeed access the TMJ disc and contribute to its degradation [MMPs contribute substantially to tissue degeneration in inflammatory joint diseases, including rheumatoid arthritis and osteoarthritis -35. Our radation .Relaxin causes the targeted induction of collagenase-1 and stromelysin-1 in synovial joint and pubic symphysis fibrocartilages but not in articular cartilage. This induction of MMPs in joint fibrocartilage is accompanied by a loss of collagen and GAGs that is prevented by an MMP inhibitor, suggesting a link between relaxin, MMPs, and matrix degradation. These studies provide the first evidence that relaxin contributes to the degradative remodeling of joint fibrocartilage and that there is an association between relaxin-induced MMPs and matrix loss; they also suggest a potential mechanism of action of relaxin in contributing to TMJ diseases in a subset of women with these disorders.The author(s) declare that they have no competing interests.TN performed all experiments, assays and analysis in which MMP inhibitors were used. TTD performed all experiments to characterize the changes in MMPs and GAGs in joint fibrocartilage in response to relaxin and β-estradiol. GH and QZ characterized the responses of the pubic symphysis fibrocartilage and articular cartilage to the hormones. MS retrieved tissues from animals and assisted in several MMP assays. SK conceived the study, participated in its design and coordination, supervised the statistical analysis, and wrote the manuscript. All authors read and approved the final manuscript.ANOVA = analysis of variance; DMMB = 1,9-dimethylmethylene blue; GAG = glycosaminoglycan; FITC = fluorescin isothycyanate; MMP = matrix metalloproteinase; PBS = phosphate-buffered saline; TIMP = tissue inhibitor of metalloproteinase; TMJ = temporomandibular joint.


It is well known that antidepressants increase neurogenesis in the dentate gyrus of the hippocampus. The increase of neurogenesis might contribute to the behavioral effects of antidepressants. However, the mechanism by which antidepressants increase hippocampal neurogenesis is largely unknown. It has been recently reported that astroglia induce the neurogenesis of the hippocampal neural progenitor cells (NPCs). Therefore, we hypothesized that antidepressants may act on astrocytes, and this in turn induces neurogenesis of NPCs.To examine this hypothesis, we used two co-culture systems, i.e., a contact-independent Banker culture and a contact-dependent overlay co-culture. In both of these systems, in comparison with naïve astrocytes, antidepressant-treated astrocytes did not further increase the proliferation of NPCs.These results suggest that astrocytes increase the proliferation of hippocampal NPCs, however, this may not be directly involved in the antidepressant-induced proliferation of NPCs. Depressive disorder is a chronic, life-threatening illness that may be caused by genetic and environmental factors such as stress . Of all It has been well known that astroglia support the functions of neurons. However, a number of recent studies have also shown that astroglia actively control many neuronal functions and development; moreover, this is related to many neurological disorders -11. ManyWe thus hypothesized that when astrocytes are activated by antidepressants, they may release factors that can induce neurogenesis in the dentate gyrus of the hippocampus. In addition, neurogenesis induced by astrocyte-releasing factors may be involved in the behavioral effects of antidepressants. To examine our hypothesis, we used two types of astrocyte-NPC co-culture systems and measured the proliferation of NPCs by using BrdU immunocytochemical analysis.Before we examined our hypothesis, we identified cultured astrocytes and NPCs by immunocytochemistry. As shown in Fig BrdU is incorporated into the DNA of proliferating NPCs. Thus, the appearance of BrdU immunoreactivity within a cell indicates that the cell was undergoing proliferation at the time upon BrdU was added. Using the BrdU-labeling method, we examined whether antidepressant-treated astrocytes could induce the proliferation of hippocampal NPCs, possibly through diffusible factors. To test this possibility, we used the Banker culture system, which does not allow direct contact between astrocytes and NPCs Fig.  17]. Th. Th17]. On the other hand, the involvement of astrocytes in the proliferation of NPCs could possibly occur in a contact-dependent manner . To test2C pre-mRNA editing responses in the forebrain [In this study, we have examined the possibility that astrocytes are involved in antidepressant-induced proliferation of NPCs. After treated with 10 μM of desipramine or fluoxetine for 48 h, astrocytes were co-cultured with NPCs in a contact-independent or contact-dependent manner. In the former case, although naïve astrocytes induced the proliferation of NPCs in comparison with NPCs that were grown alone, desipramine or fluoxetine treatment of astrocytes did not further increase the proliferation of NPCs Fig. . In addiorebrain . Second,orebrain . Althougorebrain ,20. ThusWe hypothesized that astrocytes might be involved in the antidepressant-mediated neurogenesis since it was well known that astrocytes induce the hippocampal neurogenesis and have serotonin or norepinephrine transporter which are targets of the many antidepressants. However, in both contact-dependent and contact-independent co-culture system, astrocytes did not further increase the proliferation of the hippocampal NPCs. Considering that astrocytes are involved in many other neurological functions, more investigations should be carefully performed to elucidate the role of astrocytes on the antidepressant-induced hippocampal neurogenesis.Primary NPCs from the hippocampus of postnatal day 1 or 2 (P1 or P2) C57BL6/J mice were prepared essentially as described previously ,22. Hipp2 tissue culture flask (SPL) in DMEM/F12 (1:1), 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS) (Gibco BRL). The medium was replaced every 5 days. When the cells became confluent (DIV 10–11), the flasks were shaken at 200 rpm and 37°C for 3–4 h, and the medium was replaced with fresh medium. Additionally, in case of the overlay co-culture, the cells were treated with cytosine arabinoside  for 72 h and then washed with phosphate buffered saline (PBS) three times before co-culture.Primary astrocytes from the hippocampus of P1 or P2 C57BL6/J mice were prepared as described previously ,22. Hipp4/cm2. On the following day, the cells were treated with 10 μM of fluoxetine (Sigma) or desipramine (Sigma) dissolved in the culture medium for 48 h; this was followed by washing with PBS three times. The concentration of each antidepressant was determined on the basis of the steady-state brain concentrations estimated from a clinical study [4/cm2 in the N2 medium containing 20 ng/ml bFGF.For the Banker culture, astrocytes were passaged on the poly-L-lysine-coated coverslips  at a density of 3.7 × 10al study . With real study . After w4/cm2. After one day, the cells were treated with 10 μM of fluoxetine or desipramine for 48 h; this was followed by three washes with PBS. After washing out the antidepressants, NPCs expressing GFP were overlaid onto astrocytes in the N2 medium containing 20 ng/ml bFGF.For the overlay culture, astrocytes were plated on the poly-L-lysine-coated glass- bottom dish at a density of 3.8 × 10Twenty-four hours after the co-culture, NPCs were treated with BrdU  (Sigma) for 24 h. In case of the overlay co-culture, NPCs and astrocytes were treated with BrdU. Finally, co-cultured NPCs were subjected to the immunocytochemical analysis. Briefly, the cells were fixed with 4% paraformaldehyde in PBS. For the detection of BrdU, the cells were treated with 1 N HCl at 37°C for 30 min and then neutralized by washing with 0.1 M sodium borate buffer. After the treatment with HCl, cells were incubated with cold PBT (0.1% Triton X-100 and 0.1% BSA in PBS) for 15 min. After blocking with a preblock solution (2% BSA and 0.08% Triton X-100 in PBS) at room temperature for 2 h, the cells were incubated overnight with the following primary antibodies in a preblock solution: BrdU , GFP , nestin , GFAP . After washing with PBT, the cells were incubated with the fluorescence-labeled secondary antibodies for 2 h. Cells were counterstained for 15 min with 10 ng/ml 4', 6'-diamidino-2-phenylindole (DAPI) (Molecular Probes) or 50 μg/ml propidium iodide (PI). Immunofluorescence was visualized using a fluorescence microscope (Carl Zeiss). Fluorescence-positive cells were quantified in 20 fields (×10) per dish either manually (overlay culture) or by using Image J software (Banker culture).Total RNA was collected using the TRIzol Reagent according to the manufacturer's instructions (Invitrogen). Reverse transcription (RT) reactions were performed using SuperScript™ III Reverse Transcriptase (Invitrogen). cDNA was synthesized from total RNA (2 μg) using a random primer (0.1 μg). One microliter of the RT reaction product was utilized as the template in the PCR with each of the following primer sets. GAT ATT GCA GCG GTT CCT GT and TTC GCA CTG TAG CAG GAA TG were used for GDNF, while TGC ACC ACC AAC TGC TTA and GGA TGC AGG GAT GAT GTT C were used for GAPDH. The number of PCR cycles was set such that it was below saturation conditions and was 30 and 23 for GDNF and GAPDH, respectively.The author(s) declare that they have no competing interests.H-GK carried out all the experiments outlined in the manuscripts, designed the studies and wrote the manuscript. SJL participated in astrocyte culture. HS participated in designing the study and writing the manuscript. B-KK supervised the experiments, participated in the interpretation of data and wrote the manuscript. All authors read and approved the final manuscript.


In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ+ gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro.Gene targeting lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside.An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant.The mutant frequency of the in vivo mediated by virus vectors and other means.The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting  If the gene targeting of somatic cells is made possible by gene transfer in vivo, it will facilitate the analysis of gene function, and provide a means of gene therapy for genetic and other diseases [Gene targeting, which is the precise alteration of genomic information by homologous recombination, has provided a powerful means of genetic analysis in microorganisms and mammalian systems . In mousdiseases .in vivo. First, its low efficiency makes it difficult to detect and analyze. A sensitive and accurate measurement system is therefore needed to detect such low-frequency events. Although there have been several reports of gene targeting in the rat liver with specifically designed oligonucleotides [in vivo  and analysis of the products revealed that homologous recombination was more frequent than non-homologous recombination. One possible reason for this high accuracy was protection of the viral DNA by the terminal protein, which is covalently attached to the ends of the viral DNA and to other viral proteins during its transfer to the nucleus and target DNA. Breaks in unprotected DNA would lead to non-homologous recombination.The second major problem with gene targeting in vitro ,8. Fujitin vitro . The genin vivo because it has a broad host-range, is easy to prepare to a high titer and only rarely integrates into the host genome by non-homologous recombination [The adenovirus is useful for gene delivery bination ,10. To dbination . Numeroubination ,13.in vivo.In the present study, we investigated gene targeting in the mouse liver using a replication-defective adenovirus vector and a transgenic mouse system Figure . AlthougThe bacteria, bacteriophages and plasmids used in this study are listed together with details of their construction in lacZ-negative bacteriophage lambda by phenyl beta-D-galactoside  selection (see below). BIK1564 was used for the growth of all bacteriophage lambda strains in this study. BIK2206 was used for confirmation of the LacZ-negative phenotype of the bacteriophage selected with p-gal using 5-bromo-4-chloro-3-indlyl-beta-D-galactose .BIK12001 was used for the titration of bacteriophage lambda and the measurement of lacZ gene  was confirmed by sequencing. pNY20 was produced by replacing the smaller SmaI-SacI fragment of pNY19 with the homologous SmaI-SacI fragment of pNY15G3.11, which carries the mutant sequence.The construction of the plasmids used in this study is detailed in e Figure  was usede Figure  was intrlacZ mutations were transferred back to lambda by homologous recombination in vivo [600 = ~0.3 in LB  containing 20 μg/ml chloramphenicol, 0.2% maltose and 10 mM MgSO4. LIA7 was adsorbed onto the cells at a multiplicity of 1.0 at 37°C for 15 minutes. The mixture was shaken at 37°C until the OD600 dropped below 0.3. One drop of CHCl3 was added to the mixture, which was then shaken for 30 seconds. The mixture was centrifuged and the supernatant was recovered. The supernatant was assayed for BIK12001 on agar plates containing p-gal as detailed below. The plaques on the p-gal plates were isolated and analyzed for the designed sequence change by restriction of the PCR products .These two  in vivo  in orderlacZ-negative bacteriophage particles were detected using positive selection [600 = 1.0 in LB containing ampicillin (50 μg/ml), kanamycin (20 μg/ml) and 0.2% maltose. The culture was centrifuged at 3,500 rpm for 15 minutes at 4°C. The pellets were dissolved into one-half the volume of LB containing 10 mM MgSO4. The bacteriophage was adsorbed onto these cells at room temperature for 20 minutes. To estimate the total number of bacteriophages, 2.5 ml molten 1/4 LB top agar , 6.4 g NaCl and 7.5 g Bactoagar per liter) was added to 0.25 ml of the mixture of cells and bacteriophages, and the entire content was poured onto a 1/4 LB plate . To estimate the number of lacZ-negative bacteriophages, 2 ml of the mixture of cells and bacteriophages, and 22 ml of molten 1/4 LB top agar containing 0.3% p-gal , were mixed and poured onto four 1/4 LB plates. The plates were incubated at 37°C for 12 hours.The election ,16. BIK1pNY56 was constructed by replacing the shorter XbaI-BamHI fragment of pHM5 by the XbaI-BglII fragment of pNY19 Figure . pAdHM4 9 plaque-forming units (PFU) of the recombinant adenovirus in 200 μl of PBS  was injected into the tail-vein of each mouse using a 30-gauge needle. AdNY56 was injected into one mouse, AdNY57 was injected into two mice and AdNY58 was injected into two mice.Female MutaMice (7 weeks old) were obtained from Covance Research Products Inc. . The MutaMice were maintained under specific pathogen-free conditions in the animal faculty of the Institute of Medical Science at the University of Tokyo, Japan. After the animals were anesthetized with Nembutal , 3 × 10lacZ-negative mutants were detected by p-gal selection as described above. Each plaque on the selective agar was recovered in 100 μl of SM buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgSO4, 100 mM NaCl and 0.01% gelatin). In order to verify the lacZ-negative phenotype, each isolate was assayed on agar with X-gal using a spot assay as follows. BIK2206 was grown in LB containing ampicillin (50 μg/ml) and tetracycline (10 μg/ml). Twice-concentrated culture (1.25 ml) was mixed with 6 ml molten LB/MM agar  and spread on agar. A 10-μl aliquot of each bacteriophage sample was spotted onto these cells. The plates were incubated overnight at 37°C. The mutant frequency was estimated by dividing the number of PFU on the selective plate  by the number of total PFU on 1/4 LB agar.Twenty-four hours after injection, the mice were sacrificed. A lobe of the liver of each animal was excised, frozen by submersion in liquid nitrogen and stored in a 1.5-ml plastic tube at -80°C. Genomic DNA was isolated from the liver tissue with phenol-chloroform and precipitated by ethanol/sodium as described in the manual for MutaMouse. Lambda bacteriophage particles were recovered from the isolated DNA by incubation with packaging extracts . The lacZ-negative lambda bacteriophage DNA from the mice was analyzed using restriction enzymes following PCR. For the lacZ-negative lambda DNA from the AdNY57-treated mouse, PCR was carried out with the primer pair LG-1 (5'-TACCGGCGATGAGCGAAC-3') and LG-2 (5'-CTCCAGGTAGCGAAAGCC-3'). The 288-bp product was purified by ethanol/sodium precipitation, digested with TfiI  ) at 65°C and analyzed using agarose electrophoresis. The mutant sequence was resistant to TfiI, while the wild-type sequence was sensitive, yielding 204 and 84 bp fragments. The primer pair Lam-1 (5'-TACTGTCGTCGTCCCCTC-3') and Lam-2 (5'-CGCAGATGAAACGCCGAGT-3') was used for the lacZ-negative lambda DNA from the AdNY58-treated mouse. The 213-bp PCR product was digested with XspI   at 37°C and analyzed using agarose electrophoresis. The wild-type sequence was resistant to XspI, while the mutant sequence was sensitive, yielding 146 and 67 bp fragments.The in vivo. The MutaMouse carries approximately 40 copies of bacteriophage lambda gt10lacZ on a chromosome [lacZ gene. The donor DNA was delivered to the liver cell nuclei by tail-vein injection of the recombinant adenovirus. Genomic DNA was isolated from the liver and its in vitro packaging allowed the recovery of the lambda genome in viable bacteriophage particles. A lacZ-negative mutant bacteriophage was selected as a plaque-former in an Escherichia coli mutant defective in the galE gene on an agar plate containing p-gal. This chemical is converted by the lacZ gene product  into UDP-galactose, which accumulates in the absence of the GalE protein to induce cell death. The ratio of the mutant plaque-formers to the total plaque-formers was used to estimate the fraction of the mutated gene. The mutant gene was further analyzed using restriction enzymes.Figure romosome ,19. The romosome . Our tarin vitro-ligation method were used to deliver the donor DNA [lacZ was inserted into the E1 deletion site of the mutant adenovirus [lacZ, while AdNY57 and AdNY58 had a point mutation in lacZ  from T to G, thereby generating the Tyr105Stop mutant. The mutant and wild-type sequences can be distinguished using the restriction enzyme XspI Figure .lacZ mutants that were predicted to be generated by the recombinant adenovirus could be selected with p-gal as follows. Bacteriophage lambda strains carrying the mutations were produced by transferring each mutation on a plasmid back to lambda through homologous recombination in E. coli . The two bacteriophage strains, lambda gt10lacZ- Tyr105Stop (LIA11) and lambda gt10lacZ- Glu461Gly (LIA15), were then used in the p-gal selection. As shown in Table lacZ showed a plaque-formation efficiency of less than 1/10,000 on the selective agar relative to that on the non-selective agar. By contrast, each of the mutant lambda strains showed similar or slightly decreased plaque-formation efficiency on the selective agar. We concluded that the expected targeted product with AdNY57 and AdNY58, if it was produced, should be selected and measured using the p-gal-selection procedure.We demonstrated that 9 PFU in 200 μl of PBS) were injected into the tail-vein of a MutaMouse. It is well established that the adenovirus genome accumulates in the liver cell nuclei after tail-vein injection [in vitro packaging. The lacZ-negative phage was detected selectively on agar with p-gal. The plaques on these selective plates were isolated and the LacZ-negative phenotype was confirmed on agar plates containing X-gal. The mutant frequency was estimated as the fraction of the lacZ-negative phage  copies of the target gene, which amount to 0.4% of the genome. This should be able to improve the sensitivity of detection of gene targeting, although the sensitivity is limited by spontaneous mutagenesis. In addition, the presence of tandem repeats might have other types of negative effect on gene targeting, as detailed below.Also, in the present system, a successful gene-targeting event would not be distinguishable in the phenotype of the mouse cell. In transgenic mice with a single copy of the mutant acZ gene , correct9 PFU of the virus [in situ hybridization revealed that, after tail-vein injection of 2 × 109 PFU, all of the hepatocyte nuclei had 1–100 copies of a recombinant adenovirus genome, with an average of 20 copies [8 PFU of a recombinant adenovirus with the lacZ expression cassette, 40% of the hepatocytes expressed beta-galactosidase [9 PFU in our experiment. (We cannot raise the titer any more because of the toxicity of the virus.) This type of information can be confirmed by Southern hybridization and fluorescence in situ hybridization.How efficient is adenovirus infection and delivery to the hepatocyte nucleus? Tail-vein injection is an established method for the delivery of adenovirus to liver cells. The average copy number of a replication-defective recombinant adenovirus genome per liver cell has been estimated as 14–28 copies using Southern hybridization after tail-vein injection of 5 × 10he virus . This co0 copies . After ttosidase . We assuin vitro varies from ~10-7–10-4 per cell [in vivo using an adenovirus vector or any other means, it will be necessary to increase the frequency of gene targeting. So how can we achieve this goal?The gene-targeting frequency with recombinant adenoviruses per cell ,28. We din vitro varies from one locus to another [The efficiency of gene targeting  another ,30. Such another . Thus, tlacZ transgene is methylated to a high degree at every CpG site [lacZ gene (9%) [Repetitive sequences are methylated in the mouse genome . IkehataCpG site . One posene (9%)  is much ene (9%) . Methyl-ene (9%) . Furtherene (9%) . This trene (9%) ,39. ReduN-ethyl-N-nitrosourea, which is a direct-acting DNA-ethylation agent, in the MutaMouse [Chromosome replication is known to stimulate homologous recombination. Partial hepatectomies in mice might stimulate liver cell proliferation and DNA replication, which in turn might stimulate recombination. Hara et al. (1999) reported that partial hepatectomies increased mutagenesis with utaMouse .2 resulted in an approximately three-fold increase in their mutual homologous recombination [cis-diamminedichloroplatinum) and mitomycin C [in vivo if they are shown to be active in mutagenesis in a transgenic-reporter mouse system. The effect of such recombinogenic damage might be much larger with replication-defective adenovirus recombinants than with replication-competent adenoviruses, because their replication-intermediates are responsible for their high recombination frequency [It might be easier to modify the donor DNA than the recipient DNA. One can generate recombinogenic damage on the donor DNA. Irradiating adenovirus particles with ultraviolet light of 1500 J/mbination . Recombiomycin C . Such agrequency ,43-45.The gene-targeting frequency is strongly dependent on the length of homology; the frequency increases as the homology length increases up to 10 kb -48. If tHere we attempted to perform gene targeting in a transgenic mouse system that allowed the sensitive detection of mutagenesis. The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 with the sensitive and PCR-independent detection system.PCR, polymerase chain reaction; PFU, plaque-forming unit; RFLP, restriction fragment length polymorphism; p-gal, phenyl-beta-D-galactoside; X-gal, 5-bromo-4-chloro-3-indlyl-beta-D-galactoseThe author(s) declare that they have no competing interests.AI carried out the injection of the recombinant adenovirus and the analysis of the mouse DNA. YN and HM constructed the recombinant adenovirus. NH injected the recombinant adenovirus to the mouse. YN constructed the experimental design as well as cloning of the part of lambda DNA from the MutaMouse genomic DNA. IK provided the original experimental idea and coordinated the experimental design. All authors read and approved the final manuscript.Bacterial strains, plasmids, bacteriophage strains and recombinant adenovirus constructs.Click here for file


In the past decade, much work has been done to dissect the molecular basis of the defence signalling pathway in plants known as Systemic Acquired Resistance (SAR). Most of the work has been carried out in model species such as Arabidopsis, with little attention paid to woody plants. However within the range of species examined, components of the pathway seem to be highly conserved. In this study, we attempted to identify downstream components of the SAR pathway in apple to serve as markers for its activation.PR-2, PR-5 and PR-8, which are induced in response to inoculation with the apple pathogen, Erwinia amylovora, but they are not induced in young apple shoots by treatment with known elicitors of SAR in herbaceous plants. We also identified three PR-1-like genes from apple, PR-1a, PR-1b and PR-1c, based solely on sequence similarity to known PR-1 genes of model (intensively researched) herbaceous plants. The PR-1-like genes were not induced in response to inoculation with E. amylovora or by treatment with elicitors; however, each showed a distinct pattern of expression.We identified three pathogenesis related (PR) genes from apple, PR-1a, PR-2, PR-5 and PR-8 from apple are not markers for SAR in young apple shoots. Two additional PR-1-like genes were identified through in-silico analysis of apple ESTs deposited in GenBank. PR-1a, PR-1b and PR-1c are not involved in defence response or SAR in young apple shoots; this conclusion differs from that reported previously for young apple seedlings.Four PR genes from apple were partially characterized.  Systemic Acquired Resistance (SAR) have been elucidated. The pathway leading to SAR involves three steps, pathogen recognition, signal relay and induction of genes, which facilitate synthesis of protective molecules. Once the pathogen is detected, the plant relays a signal through a complex network of signalling molecules to transcription factors that activate transcription of defence proteins or production of secondary metabolites [Pathogenesis-Related (PR) proteins have been used routinely in studies with model (intensively researched) species to assess the defence status of plants.Botanists have known for nearly 100 years that plants, like animals, can be immunized against pathogen attack by pre-treatment with another pathogen [abolites . Some doArabidopsis thaliana in response to the oomycete pathogen Peronospora parasistica differs from the suite induced in response to the fungus Alternaria brassicicola [PR-proteins of plants have been defined as proteins of a host that are induced only in response to attack by pathogens or by a related event . PR protsicicola . The presicicola .Arabidopsis thaliana [Solanaceae family, including tomato and tobacco [PR-1, 2, 5, and 8 is characteristic of SAR in several herbaceous plants. In tobacco, PR-1 protein can account for 1% of the total leaf protein in TMV-infected tissue [PR-8 is robustly induced following treatment with SA or the related, but less phytotoxic compound INA , both of which induce SAR [There is a plethora of information about SAR and PR genes related to several model plants, especially, thaliana , and mem tobacco ,7. In or tobacco . Inductid tissue . In cucuduce SAR .Very little molecular evidence for SAR in woody perennials has been reported. Several groups have reported phenotypic resistance to pathogens following application of SAR elicitors such as SA or its functional analogs; benzothiadiazole-7-carbothioic acid-S-methyl ester (ASM) and INA to apple and pear -14. HoweE. amylovora, which causes the devastating disease known as fire blight [We undertook this study in an attempt to identify markers for the SAR pathway in apple. Specifically, we assayed apple tissue for induction of homologues of known PR genes following inoculation with the bacterial pathogen e blight . In addiPR genes from apple were identified through a degenerate primed PCR approach with a cDNA library of Malus × domestica cv. Gala. The library, used as template in PCR, was developed from a pool of young apple shoots harvested from 0 to 6 days after inoculation with E. amylovora strain Ea273. Southern blot analysis of apple genomic DNA using the protein encoding regions of PR-1a, PR-5 and PR-8 from Malus × domestica cv. Gala as probes revealed that the three putative PR genes identified in apple, like those in other species, are members of multi-gene families. The full-length probes hybridized to multiple bands under high stringency conditions. Comparison of the predicted apple gene product to the type member for each group, as described by Van Loon et al[The protein coding portions of three The predicted gene products were analyzed for putative sub-cellular localization using PSORT, version 6.4, on the ExPASy Proteomics Server . Apple PPR-1a, PR-5 and PR-8 were deposited in GenBank [DQ318212, DQ318213 and DQ318214, respectively.The nucleotide sequences of apple  GenBank , and thePR-1 family in apple. Three distinct groups of EST's were found based on predicted amino acid sequence similarity. The groups were arbitrarily designated PR-1a, PR-1b and PR-1c. An alignment of the three genes with the type member (tobacco PR-1a) is shown in Fig. An in-silico analysis of apple ESTs deposited in GenBank was carried out to identify other members of the PR-1 genes, the predicted protein product of PR-1a is most similar to the type member, tobacco PR-1a. Furthermore, PR-1a is the only PR-1 protein from apple reported to date that is predicted by PSORT to have a cleavable N-terminal signal sequence and to be localized outside of the cell (score = 0.820). PR-1c is predicted to contain an un-cleavable N-terminal signal sequence and to be localized to a membrane . PR-1b is predicted to be a cytoplasmic protein (score = 0.650).Of the three different apple PR-1b and PR-1c sequences was either fruit or flower tissue. In contrast, ESTs corresponding to PR-1a came from diverse sources; fruit (GenBank: CO576594), flower (GenBank: CO419366), shoot internode (GenBank: CV630152), leaf (GenBank: CV524932), bud (GenBank: CO903582) and even plantlets grown in-vitro (GenBank: AF507974)  or mock inoculation. Digoxigenin-labelled probes covering the entire open reading frames of PR-5 and PR-8 were used. In addition, a digoxigenin-labelled fragment of apple PR-2 (GenBank: AY548364) also was used as a probe. Expression levels were followed from pre-inoculation through 96 hours post-inoculation. PR-2, PR-5 and PR-8 were robustly induced in apple shoots between 24 and 48 hours post-inoculation with E. amylovora, but expression of PR-2, PR-5 and PR-8 was not induced in either mock-inoculated or P. syringae-inoculated apple shoots .In contrast to the robust induction of 3000 Fig . The exp®, as determined by northern hybridization analysis  per liter proved phytotoxic to apple leaves and shoots within 48 hours after spray application.None of the four apple PR genes identified here were induced during the first 96 hours following treatment with ASM or ProActsis Fig. . Subtle E. amylovora based on their similarity to genes documented as involved in SAR in other plants. Three of the four apple genes, PR-2, PR-5 and PR-8, but not PR-1a, conform strictly to the definition of a PR gene described by Van Loon et al. [E. amylovora.We identified four genes as candidates for involvement in the response of apple to attack by n et al. ; they arPR-1a was not induced following inoculation with the apple pathogen, E. amylovora. Based on work in Arabidopsis, tobacco and other species [E. amylovora, PR-1a falls short of meeting the strict definition of a PR gene, and may be more properly referred to as a "PR-like" gene.We were surprised that  species , we expePR-1b and PR-1c diverge significantly from PR-1a in the highly conserved fourth alpha helix region. They are expressed in distinctive patterns during flower development; they were not expressed in apple shoots whether or not the shoots were inoculated with E. amylovora. This is an interesting observation, which raises the question as to the possible involvement of PR-1b and PR-1c in floral development.The other two members of the PR-1 gene family identified here, PR-1 gene family exists in apple, which is up-regulated during pathogen interactions, a recent report by Gau et al. [PR-1 is not induced in apple shoots during pathogen attack. These authors analyzed the protein content of apoplastic fluid of the apple cultivar Elstar following inoculation with Venturia inaequalis, the apple scab pathogen. They did not detect any PR-1-type protein up to 21 days following inoculation. Thus, for at least two apple pathogens, E. amylovora and V. inaequalis, PR-1 is not part of an induced defence response in shoots for at least the first 96 hours and 21 days following inoculation, respectively.Although we cannot rule out the possibility that an unidentified member of the u et al.  seems toE. amylovora, with 10 mM SA or ASM at 200 mg AI per liter [E. amylovora 10 days later. There was a significant reduction in disease incidence and severity in treated trees. However, expression of PR-1 was not affected by treatment of pear shoots with ASM or SA or following inoculation with E. amylovora; the authors concluded that PR-1 was expressed constitutively in pear shoots and was likely not involved in SAR in pear [In 2004, Sparla et al. reported a study in which they had treated pear trees, another important host of er liter . Trees w in pear .PR-10 in apple seedlings following spraying with ASM at 200 mg AI per liter [® at 250 mg AI per liter to apple shoots growing on mature wood did not result in significant induction of the four PR genes assayed . The dose of Acitigard® used in this study was well within the range used by others, and is more than 10 times the application rate recommended in the product literature [Several other groups have reported increased resistance to the development of fire blight in host plants treated with ASM ,12,14. Mer liter . In eachPR-2 we observed following treatment with INA at 250 mg AI per liter was a wound response coincident with the development of phytotoxicity.Our studies of PR gene expression in shoots following treatment of 1-year-old apple trees with elicitors do not support the conclusion that induction of the SAR pathway is responsible for the phenotypic increase in resistance to fire blight reported by others ,12,14. IE. amylovora with respect to extent and rate of symptom development and for induction of PR gene expression. The florist's frog method is similar to a method used by van der Zwet and Keil [E. amylovora by apple cells.We evaluated three methods for inoculating shoots of 1-year-old apple trees with and Keil , but it and Keil , was incPR-2, PR-5 and PR-8 was apparent in apple shoots 24 to 48 hours after inoculation with E. amylovora, the fire blight pathogen. Enhanced expression of PR-2, PR-5 and PR-8 was not observed when apple shoots were inoculated similarly with P. syringae pv. tomato, a non-pathogen of apple.Enhanced expression of PR-1a in apple shoots was not enhanced during the first 96 hours after inoculation with either E. amylovora or P. syringae pv. tomato, nor was PR-1a expression induced in response to treatment with compounds known to elicit SAR in other plants. Thus, we conclude that PR-1a, PR-1b and PR-1c are not involved in defence response or SAR in young apple shoots; this conclusion differs from that reported previously for young apple seedlings.The expression of Treatment of apple shoots with elicitors of SAR in other plants did not result in enhanced expression of any of the four PR genes identified in apple. Thus, we were not able to identify markers for SAR in apple.E. amylovora was effective in inducing expression of PR genes; symptom development occurred rapidly following inoculation with the florist's frog.Inoculation of apple shoots with the pins of a florist's frog contaminated with cells of Malus × domestica cv. Gala trees were planted in soil mix  : 1 part Perlite with Osmocote  in 3.8-liter pots and placed in the greenhouse. Trees were trained to two shoots. When shoots were 20–30 cm long, the trees were transferred to a controlled environment chamber where they were maintained at 24°C – 26°C with a 12-hour photoperiod (380 μM/m2s incandescent and fluorescent) and a minimum relative humidity of 65% for the remainder of the experiment. Trees were given a 3 – 4 day equilibration period in the growth chamber prior to further manipulation.Dormant 1-year-old Apple flowers, staged according to Chapman and Catlin , were haErwinia amylovora strain Ea273 or P. syringae pv. tomato (DC3000) were grown for 16 hours at 26°C on plates of Luria-Bertani (LB) medium. Colonies were transferred to 5 mM potassium phosphate buffer, pH 6.5, using a cotton swab. The density of the suspension was adjusted to O.D.600 = 0.2, which corresponded to 108 cells/ml. Unless mentioned otherwise, inoculations were performed between 2 and 4 hours into the light cycle by dipping a florist's frog (4.8 cm in diameter with 127 pins) into freshly prepared inoculum and then puncturing the fanned-out shoot tip held against a nitrile-gloved hand. The dip and puncture procedure was repeated once. Mock inoculation was similar except that 5 mM potassium phosphate, buffer pH 6.5 was used rather than bacterial suspensions. For the inoculation optimization study, the first two unfolded, but unexpanded leaves, of ten shoots of apple trees were cut either perpendicular or parallel to the mid-vein with scissors or were punctured twice with the pins of a florist's frog dipped in inoculum. Two shoots representing each inoculation method were collected at each time point.® , was applied at 250 mg AI per liter. ProAct®  was applied at 15 mg AI per liter.Elicitors were sprayed to run-off using a hand-pumped atomizing sprayer. Elicitors were diluted in water and were applied 2 to 4 hours into the light cycle. INA was applied at 250 mg AI per liter. ASM, as Actigard® RNA Assay Kit, as directed by the manufacturer, .Harvested apple shoots were frozen by plunging the excised portions into liquid nitrogen. Once frozen, the tissue was stored at -80°C. RNA was isolated from the leaf tissue as described by Komjanc et al. , then quPR-2 (GenBank:AY548364), or the entire open reading frames of apple PR-1a, PR-5 and PR-8  following the LD PCR protocol. The full-length open reading frame  amplicons were cloned into pBluescript II KS+  and sequenced. PCR was carried out using either Pfu Turbo® (Stratagene) or DyNAzyme™ EXT  DNA polymerase, dNTP's (Promega), primers . An annealing temperature of 55°C was used for all primer sets except PR-1b; primers were given 1 minute per kb amplicon for extension at 72°C. An annealing temperature of 50°C was used for PR-1b. Cycle number was optimized for each template and primer combination, as noted in the figure legends.Degenerate primers were designed based on alignment of several known PR gene sequences deposited in GenBank. First, the degenerate primers were used to amplify putative PR gene fragments from genomic Eco RV or Hind III, resolved on an agarose gel and transferred to uncharged nylon membranes [Genomic DNA was isolated from three cultivars of apple – Jonagold, Gala and Roger's Mac, using the procedure described by Dellaporta et al. . Ten micembranes . MembranTwo micrograms of total RNA were reverse transcribed as described by Wilson and Melton , except ® from DNASTAR . Protein localization prediction analysis was run through PSORT [DNA sequence analysis, protein deduction, statistics and alignments were generated using Lasergenegh PSORT JMB carried out the experiments, conducted the in-silico analyses, prepared the figures and drafted the manuscript. JFK was instrumental with degenerate primer design and manuscript revision. SVB was responsible for experimental design and revised and polished the manuscript. All authors have read and approved the final manuscript.


PS-I is a chlorophyll-containing membrane protein complex that is the primary reducer of ferredoxin and the electron acceptor of plastocyanin. We isolated the complex from the thylakoids of spinach chloroplasts using a chemical detergent. The chlorophyll molecules associated with the PS-I complex provide an intrinsic steady-state emission spectrum between 650 and 800 nm at −196.15 °C that reflects the organization of the pigment-protein interactions. In the absence of detergents, a large blue shift of the fluorescence maxima from approximately 735 nm to approximately 685 nm indicates a disruption in light-harvesting subunit organization, thus revealing chlorophyll−protein interactions. The commonly used membrane protein-stabilizing detergents, N-dodecyl-β-D-maltoside and N-octyl-β-D-glucoside, only partially stabilized the approximately 735-nm complex with approximately 685-nm spectroscopic shift. However, prior to drying, addition of the peptide detergent acetyl-AAAAAAK at increasing concentration significantly stabilized the PS-I complex. Moreover, in the presence of acetyl-AAAAAAK, the PS-I complex is stable in a dried form at room temperature for at least 3 wk. Another peptide detergent, acetyl-VVVVVVD, also stabilized the complex but to a lesser extent. These observations suggest that the peptide detergents may effectively stabilize membrane proteins in the solid-state. These designed peptide detergents may facilitate the study of diverse types of membrane proteins.We used a class of designed peptide detergents to stabilize photosystem I (PS-I) upon extended drying under N A designer peptide detergent may facilitate the study of diverse types of membrane proteins. However, many detailed structures of diverse membrane proteins remain elusive. Thus the discovery and design of new detergents are acutely needed to facilitate membrane protein crystallization and structural studies [The bioinformatics and computational analyses of a large number of completely sequenced genomes suggest that over 30% of all genes encode membrane proteins –4. These studies .Photosystem I (PS-I) is a membrane protein complex that is associated with electron transport and exists as a large, multisubunit complex with dozens of transmembrane-spanning domains ,7. The cIn order to fabricate protein-based devices, it is crucial to immobilize and stabilize the selected proteins and enzymes for an extended time. Although some progress has been made –11, litthttp://www.anatrace.com/). In some cases these detergents have partly facilitated selective solubilization of membrane proteins from their native environments. Recent evidence suggests, for example, that detergents with short alkyl chains are more effective at solubilization than those with long alkyl chains that often lead to protein denaturation [The difficulties associated with solubilization and stabilization of membrane proteins are largely due to exposure of their hydrophobic domains, which are normally buried within the lipid bilayer. These hydrophobic transmembrane segments render the protein particularly difficult to stabilize after removal from the membrane. In particular, they are extremely susceptible to aggregation in aqueous media. In previous decades, a wealth of chemical detergents was synthesized for the study of membrane proteins , is not only able to preserve the original spectral properties of the PS-I complex, but also stabilizes PS-I in the dry form for a period of at least 3 wk. In light of the recently reported pea PS-I crystal structure [We here report the stabilization of spinach PS-I complex following chemical detergent isolation. We studied the ability of these peptide detergents to interact with and stabilize isolated spinach PS-I complexes that have been dried under Ntructure , we alsoTo evaluate the effect of the peptide detergents for stabilizing membrane proteins, we isolated the chlorophyll pigment protein from spinach chloroplast thylakoids. PS-I was isolated from spinach using a modified procedure of Mullet and Arntzen . The typFor PS-I complex dried in the absence of peptide detergent, a significant change in the fluorescence spectrum was observed. This change consisted of a decrease in the overall intensity of the far-red fluorescing chlorophyll in the approximately 735-nm region and an associated blueshift of this peak. In addition, the fluorescence peak at approximately 680 nm demonstrated an overall increase in intensity after the drying process. In some cases, a blueshift of this peak was also observed. The emission spectra of dried and solution state samples in the present chemical detergents are shown in We used four widely known membrane protein-stabilizing detergents, Triton X-100, N, N-dimethyldodecylamine N-oxide, N-dodecyl-β-D-maltoside (DM), and N-octyl-β-D-glucoside (OG) for the PS-I complex. Triton X-100 and N, N-dimethyldodecylamine N-oxide did not demonstrate a stabilizing effect based on the emission spectrum of PS-I, but in most cases caused increased degradation of the complex (data not shown). On the other hand, OG and DM showed modest stabilizing effect at low concentrations. The spectra for OG and DM are shown in 6K  and acetyl-VVVVVVD (V6D)  could stabilize PS-I complex during the drying process. Samples were prepared by addition of A6K or V6D at concentrations of 0.016%, 0.031%, 0.062%, 0.125%, 0.25%, and 0.5% . The results are shown in 6K or V6D, PS-I not only showed blueshift but also showed a reduced fluorescent emission after drying. On the other hand, at higher concentration, the PS-I complex appeared to be stabilized during and after drying. In particular, samples containing 0.5% A6K demonstrated essentially negligible change in their emission spectra in the approximately 735-nm region. V6D was less effective at stabilizing PS-I as shown in We carried out systematic experiments to determine if the peptide detergents A6K could stabilize the PS-I complex on a dry surface. The stability of dried PS-I with 0.5% A6K (34X CAC in PBS) on a glass slide was measured at days 1, 7, 14, and 21. The results are shown in 6K peptide detergent is able to stabilize the PS-I complex under the conditions used.We asked how long the peptide detergent AThis observation is consistent with a separate experiment of another membrane protein, rhodopsin, using other peptide detergents. In these experiments, rhodopsin was stabilized for an extended time when high concentrations of peptide detergents were used .Study of the low-temperature chlorophyll fluorescence in the approximately 735-nm region was originated over 25 y ago . It was The ability to monitor the reversible dissociation of pigment–protein complexes and the consequent cessation of intercomplex energy transfer by the relative intensities of −196.15 °C fluorescence at 685 nm and 735 nm has long been realized . MonitorStudies have also been carried out on the −196.15 °C emission spectrum of the PS-I core. Based on these studies, in which there is significant fluorescence at approximately 715 nm, it is presumable that damage could be done to the PS-I during drying as well. It is interesting to note that similar results have been reported from adverse interactions with detergent . In thesMuch progress has been made in the study of PS-I, mostly through the combination of membrane fractionation studies, various types of quantitative spectroscopy, protein crystallography and structural analyses. The chlorophyll pigment proteins responsible for these various spectrally distinct species are finally being resolved ,7. The −lhca1–4 genes showed that the approximately 730-nm emission could be abolished with a commensurate increase in the fluorescence at approximately 680 nm [The spectral changes presented here could therefore be the result of changes in the LHCI peptides, or in their association with the PS-I core complex. Several experiments on LHCI peptides have given similar results to those presented here. For example, it was shown that heterodimerization was an essential feature of LHC-730 . The fluy 680 nm  by altery 680 nm ,47. ThesThe current understanding for chlorophyll distribution in native PS-I including the antennae LHCI is that the core complex has approximately 110 chlorophyll molecules associated with PsaA/B. The peripheral antennae contain approximately 84 chlorophyll molecules composed of four distinct subunits, LhcA1–4, that exist in the form of heterodimers. These heterodimers are isolated as two fractions: LHCI-A (composed of LhcA1 and LhcA4) and LHCI-B (composed of LhcA2 and LhcA3). From analysis of chlorophyll content of isolated complexes and reconstituted complexes, each LHCI monomer subunit appears to bind approximately 10 chlorophyll molecules. This would then suggest that the native complex with approximately 195 chlorophyll molecules must have four heterodimers associated with each core complex. Undoubtedly, the precise number of antenna LHCI subunits will vary with plant species, plant age, and environmental conditions. In fact, analysis of the recently solved crystal structure of pea PS-I, which contains only 167 chlorophyll molecules, reveals that there are only two heterodimers. These four LHCI subunits that are asymmetrically positioned on one side of the PS-I core complex form an “LHCI belt” .2+–Mg2+ distances between the core complex chlorophyll molecules and the primary LHCI chlorophyll molecules would suggest that energy transfer between the LHCI belt and core complex is directed through specialized “gap” chlorophyll molecules at the junction of LhcA1 and subunit G . In collaboration with another investigator, using the peptide detergent V6D, a crystal of the integral membrane protein glycerol 3-phosphate dehydrogenase with its cofactor was obtained and was diffracted to approximately 7 Å using an in-house X-ray diffraction instrument .PS-I is one of the first examples that clearly demonstrate how designed peptide detergents can stabilize membrane proteins. In collaboration with others, we have also stabilized bovine rhodopsin with the peptide detergents Atrans-membrane domains of membrane proteins, thus sequestering them from directly interacting with water molecules and preventing them from undergoing self-aggregation. In the PS-I complex, the peptide detergents not only sequestered the protein and filled the “gaps” but also maintained its overall structural integrity, thus retaining its biological function [It is plausible that peptide detergents, similar to other detergents, may directly interact with the hydrophobic domains of membrane proteins. It is likely that numerous peptide detergent molecules, approximately 2 nm in size, like lipids, can effectively surround the hydrophobic function .The applications of designed peptide detergents are still in an early stage of development. The peptide detergents presented here represent a new class of bioengineered material because they can be designed at the molecular level, using a combinatorial approach characteristic of peptide chemistry. However, we have yet to introduce the variability inherent in amino acids. For example, so far, we have concentrated on peptides containing one kind of hydrophobic tail. It is likely that as we explore combinations of short and long tails, as well as different head groups, the molecules will become more specialized, and we may find specific sequences that work best for stabilizing specific membrane proteins. Equally useful is the exploration of mixing several different detergents together as a cocktail. In essence, this could let the protein select the most appropriate mixture for stabilization and crystallization.http://www.anatrace.com/) Anapoe Triton X-100. Gentle stirring began immediately at room temperature for 30 min. Following the incubation, the solution was spun at 42,000 × g for 30 min, and the pellet was discarded. Approximately 8 ml of the supernatant was loaded onto approximately 25 ml linear 0.1–1 M sucrose gradients containing 0.02% Triton X-100 with 2 ml of 2M sucrose (no Triton) as a cushion and spun at 100,000 × g in a Beckman SW-28 rotor overnight (15 h). The dark green band at the 1M–2M interface was harvested with a long needle and passed through a Dounce homogenizer, pooled, and frozen at −80 °C. The protocol was adapted from Mullet et al. [PS-I was obtained from spinach leaves by homogenization in 400 mM sorbitol and 50 mM tricine-KOH (pH 7.8). The resulting homogenate was then passed through Miracloth , supported by a double layer of cheesecloth, and centrifuged at 1,000 × g for 5 min. The pellet, consisting mostly of chloroplasts and broken plastids, was resuspended and forced through five passes in a Dounce homogenizer in 50 mM sorbitol, 5 mM EDTA-NaOH, and 50 mM tricine-KOH (pH 7.8), which lysed any unbroken plastids. It was centrifuged again at 10,000 × g for 5 min; this step was repeated once. The resulting pellet was highly enriched in thylakoids, which were completely nonappressed after treatment with the chelator; this pellet was resuspended in a small volume of water, and the chlorophyll content was determined. The thylakoid suspension was then brought to a final concentration of 0.8 mg/ml chlorophyll and 0.8% (w/v) Anatrace . The peptide detergent stock solutions were made by dissolving these peptides at a concentration of 1% (w/v) in 50 mM Tris (pH 7.8); 25 mM NaCl; 0.02% Triton X-100, and then sonicating in a water bath sonicator for 20 min. Stock solutions of peptides were stored at room temperature for the duration of the experiment.Peptides were obtained from the MIT Biopolymers Laboratory and Synpep Corporation .The structure of pea PS-I was determined at 4.4 Å . We highhttp://www.rcsb.org/pdb/) accession number for the crystal structure of pea PS-I is 1QZV.The Protein Data Bank (


The main purpose of this study was to investigate the impact of long-term endurance training on the immune and endocrine system of elderly men. The possible interaction between these systems was also analysed.elderly runners showed a significantly higher T cell proliferative response and IL-2 production than sedentary elderly controls. IL-2 production was similar to that in young adults. Their serum IL-6 levels were significantly lower than their sedentary peers. They also showed significantly lower IL-3 production in comparison to sedentary elderly subjects but similar to the youngs. Anabolic hormone levels did not differ between elderly groups and no clear correlation was found between hormones and cytokine levels.highly conditioned elderly men seem to have relatively better preserved immune system than the sedentary elderly men. Long-term endurance training has the potential to decelerate the age-related decline in immune function but not the deterioration in endocrine function. Human immune function undergoes adverse changes with aging, potentially leading to an increased risk of infections, a greater occurrence of autoantibodies and lymphoproliferative disorders, and a greater morbidity and mortality in the elderly . Of the The endocrine system also suffers from senescence . SubstanThe question arises whether regular physical activity can correct the deleterious effects of aging on the human immune and endocrine systems. There is growing evidence that long-term conditioning may be associated with improved immune functioning in the elderly , and phyThe purpose of the present investigation was to examine the effects of long-term endurance training on the T cell proliferative response, broad cytokine profile and serum hormone levels in elderly men and to correlate the anabolic hormones with cytokines.Preservation of immunological functions is keenly needed to avoid disease and consequently for the improvement of quality of life in the elderly. Several methods to improve the immune system have been tested but their effectiveness is generally controversial . Among tin vitro T cell responses to polyclonal stimulation when compared to sedentary elderly subjects  thymidine  18 hours prior to harvesting, and the amount of radioactivity incorporated was determined with a scintillation counter . Proliferation values are presented in the form of stimulation index, calculated from the ratio radioactivity in stimulated culture to radioactivity in non-stimulated culture.PBMC suspensions were prepared from heparinized venous blood using a Ficoll-Hypaque gradient, washed with RPMI 1640 medium, and resuspended in RPMI supplemented with 10% type AB human serum . Two × 10Culture supernatants were harvested by centrifugation after stimulation with PHA for 24 h, as described above. Supernatants were stored at -70°C until analysis in duplicates using a solid-phase sandwich ELISA kit .Levels of IL-2, IL-3, IL-4, IL-6, IL-10 and IL-12 were measured. Results were expressed as pg/ml. The detection limits were: <0.7 for IL-2, <7.4 for IL-3, <10 for IL-4, <0.7 for IL-6, <3.9 for IL-10 and <5.0 for IL-12.Serum cytokines levels were also determined in elderly subjects.Dehydroepiandrosterone sulfate was assayed by radioimmunoassay and growth hormone was assayed by immunofluorometric assay. Testosterone was assayed using an electrochemiluminescence immunoassay and free testosterone value was calculated from total testosterone and immunoassayed SHBG concentrations . Cortisol was assayed using a fluoroimmunoassay. The intraassay and interassay coefficients of variation were below 7.5% and 8.0% in each test, respectively.Serum hormone levels were measured only in elderly subjects.post-hoc multiple comparisons, a Tukey test or a Dunn procedure were made. Comparisons of the values between two groups were performed by Student t-test or Mann-Whitney rank sum test depending on the normality of the distribution curves. The correlation between two variables was analyzed by the Pearson correlation coefficient. The level of significance was set at p < 0.05.Statistical analysis and power calculations were performed using the SigmaStat software . Results are expressed as mean ± SEM. Group comparisons were made using a one-way ANOVA or Kruskal-Wallis test. For


BCG-vaccination can confound tuberculin skin test (TST) reactions in the diagnosis of latent tuberculosis infection.Mycobacterium tuberculosis specific whole blood interferon-gamma assay  during ongoing investigations among close contacts of sputum smear positive source cases in Hamburg, Germany.We compared the TST with a During a 6-month period, 309 contacts (mean age 28.5 ± 10.5 years) from a total of 15 source cases underwent both TST and QFT-G testing. Of those, 157 (50.8%) had received BCG vaccination and 84 (27.2%) had migrated to Germany from a total of 25 different high prevalence countries . For the TST, the positive response rate was 44.3% (137/309), whilst only 31 (10%) showed a positive QFT-G result. The overall agreement between the TST and the QFT-G was low , and positive TST reactions were closely associated with prior BCG vaccination . In contrast, there was good agreement between TST and QFT-G in non-vaccinated persons , increasing to 0.68 (95% CI 0.46–0.81), if a 10-mm cut off for the TST was used instead of the standard 5 mm recommended in Germany.M. tuberculosis-specific antigen should be recommended as a basis for the decision on whether to perform subsequent chest X-ray examinations or to start treatment for latent tuberculosis infection.The QFT-G assay was unaffected by BCG vaccination status, unlike the TST. In close contacts who were BCG-vaccinated, the QFT-G assay appeared to be a more specific indicator of latent tuberculosis infection than the TST, and similarly sensitive in unvaccinated contacts. In BCG-vaccinated close contacts, measurement of IFN-gamma responses of lymphocytes stimulated with  Mycobacterium tuberculosis (MTB) by applying classical epidemiological and molecular strain-typing techniques [Even in countries with a low tuberculosis (TB) incidence, population-based studies have recently revealed a high frequency of transmission of chniques -5. RoutiMycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine strain, the most widely used vaccine ever, and non-tuberculous mycobacterial (NTM) species. Therefore, alternative diagnostic tools for the detection of LTBI have been explored. Two proteins from M. tuberculosis, ESAT-6 and CFP-10, stand out as suitable antigens that induce a strong IFN-γ-secreting CD4 T-cell-mediated immune response to infection [M. bovis BCG and most NTM's, with the exceptions of M. szulgai, M. marinum, and M. kansasii.The tuberculin skin test (TST) introduced by Mantoux has been widely used as the screening test of choice to identify individuals with LTBI for more than a century. One of its intrinsic problems, however, is its cross-reactivity with antigens present in other mycobacteria, such as the nfection  and are ®-TB Gold In Tube (QFT-G), employs whole blood collected and incubated with overlapping peptides representing the TB antigens ESAT-6 and CFP-10 along with a peptide from another TB-specific antigen, TB7.7 (Rv 2654). For this version of QFT-G, evacuated tubes are pre-coated with control and test antigens and the blood-collection tubes also serve as the incubation vessels. The QFT-G test measures the amount of IFN-γ produced by Tcells previously exposed to MTB when they are stimulated with the TB-specific antigen during overnight incubation. Whereas the sensitivity of the QFT-G assay appears at least comparable to that of the TST for the detection of active MTB disease [A new diagnostic test for LTBI, QuantiFERON disease ,9, the s disease ,10. Howe disease , or in s disease ,13. UntiThe city of Hamburg  is particularly affected: in 2004, Hamburg had a TB incidence rate of 12.0 per 100,000. This rate is higher than in any of the other fifteen German federal states, and in recent years, against the national downward trend, it has been rising . Hamburget al. [Close contacts of sputum-smear-positive source cases were prospectively enrolled into the study over a 6-month period from May 1st to October 31st 2005. "Close" contacts were defined as household and intimate contacts; these also comprised employees who had demonstrable continuous exposure to the source case, or pupils sharing the same classroom. According to the definition of Behr et al.  in any cEach individual was interviewed by trained public-health staff using a standardized questionnaire. Information was obtained on: the contact's sex, date and country of birth, nationality, immigration status (if applicable), number of weeks of residence in Hamburg , current address (or whether the contact was homeless), the nature of the contact's current employment (if any), the nature of contact to the source case, the time interval between the most recent suspected exposure date and the date of diagnosis of the source case, details of any previous contact tracing, results of previous tuberculin skin testing and chest radiographic findings, BCG vaccination status , and associated medical problems .The QFT-Gin-tube methodology  involves two processes: (1)collection of whole blood into an evacuated tube containing TB-specific antigens and a control tube, and incubating overnight, and (2) measurement of IFN-γ production by ELISA in harvested plasma. Venous bloodwas collected from each subject before administration of Mantoux TST intotwo heparinised blood tubes calibrated to draw 1 ml of blood. The control tube contained onlyheparin, asa negative control; the heparinised TB antigentubecontained dried overlapping peptides representing the entire sequences of ESAT-6 and CFP-10 and a peptide from the TB antigenTB7.7 . The tubes were shaken and immediately incubated at 37°C for16–24 hours, after which they were centrifuged and plasma harvested. Plasma was kept refrigerated at 4–6°C until the ELISA was performed. The IFN-γ ELISA was performed using the method recommended by the manufacturer . IFN-γ vvice-versa.For the TST, 0.1 ml of tuberculin 10 GT Behring  was injected intradermally into the volar aspect of the forearm and transverse induration diameter was measured 72 hours later. Individuals performing the TST were blinded to QFT-G results and p values reported are based on two-tailed comparisons, with statistical significance set at p < 0.05.Categorical data were compared by the χ-squared test . The Wilcoxon rank sum test was performed to determine whether the distribution of continuous variables differed between two groups. Concordance between the results of the TST and QFT-G tests was assessed by using κ coefficients, both for contacts with and without BCG vaccination. Kappa values below 0.4 indicate weak correlation, values of 0.41–0.60 indicate good agreement and values above 0.6 strong agreement . LogistiThe contact investigations yielded a total of 311 persons with identified risk of LTBI due to their exposure to sputum-smear-positive source cases. Two contacts did not come back for their TST to be read and were not used in the final analysis. Thus, 309 contacts of a total of 15 different sputum-smear-positive source cases formed the study population, the demographic and clinical features of which are described in Table The number of close contacts per source case varied between 1 and 39 individuals; the mean exposure time (± SD) was 221 (± 273) hours, with a range from 40 to 1004 hours (the latter corresponding to 42 days).n = 160, 51.8%) than male. Only 8 contacts had previously been involved in contact tracing or in an employment-related investigation, and of these only one had given a positive TST result at that time (and was TST-positive as well as QFT-G -positive in the present study).Of the 309 contacts, 225 (72.8%) were born in Germany, while 84 (27.2%) had migrated to Germany from a total of 25 different countries. All of the migrants came from high-incidence MTB countries , includp<0.001) of those born in Germany.Overall, 137 of the 309 contacts (44.3%) developed an induration greater than 5 mm at the TST site and of these 28/137 20.4%) were positive by QFT-G, whereas 3 of the 172 (1.7%) who had a negative TST result were QFT- G positive . Fifty of the 84 (59.5%) foreign-born had been previously BCG-vaccinated, compared with 107/225 (47.6%) of those born in Germany (n.s.). For all positive induration sizes (i.e. >5 mm), the mean induration size (± SD) of the TST was 11.2 (± 4.3) mm in the BCG-vaccinated TST-positives and 12.1 (± 4.9) mm in the unvaccinated contacts, but this difference was not statistically significant. Thirteen contacts (4.2%) had an induration size of 5 mm and just failed to reach a positive result (= 6 mm), and 16 (5.2 %) had a response of exactly 6 mm. However, there were only two QFT-G results  close to its cut off of 0.35 IU/ml: Most of the negative results were 0.0 (n = 138) or nearly 0, the mean of positive results was 7.37 IU/ml (ranging from 0.38–21.43 IU/ml).Of all contacts, 157 of 309 (50.8%) had received BCG vaccination  were positive in both tests  were QFT-G-positive.In contrast to the high rate of TST positivity as described above, only 31 of the 309 contacts (10%) showed a positive QFT-G result. For those with a history of BCG vaccination, none of the 47 TST-negative contacts were QFT-G positive, and 14 of 110 TST-positive contacts , with concordant results in only 197/309 (63.8%). Broken down by TST result, this corresponded to concordance in 169/172 (98.3%) contacts with TST-negative results, but only 28/137 contacts (20.4%) with TST-positive results. Concordance between the two tests was poor in those BCG vaccinated , but was high  in those who had not been vaccinated Figure . There wWe also examined the possibility that adopting a higher cut off point for positivity in the TST might also result in higher levels of agreement between the two tests. Following the current guidelines of the American Thoracic Society , we raisp < 0.0001) in BCG-vaccinated contacts (Table p < 0.0001). At a cut off of 10 mm (Table p < 0.0001). The QFT-G test results, however, were only associated with foreign origin as an independent predictor  were nearly 13 times higher  indicating the likelihood of a false positive reaction due to cross-reactivity with M. bovis BCG strains. Secondly, we found that more than one-half of the contacts  had previously been BCG vaccinated, and this was independent of their origin (German or foreign-born) and their age, because even in Germany BCG vaccination was recommended up to March 1998.Since the TST is still regarded by many as the gold standard for the diagnosis of LTBI, official guidelines ,21 curreOf note was the finding that persons who were BCG vaccinated and QFT-G-positive were more likely to have TST responses >10 mm than those QFT-G-positive, but unvaccinated. This suggests that BCG vaccination plays an important role in the routine immunological memory of MTB infection, and may prime the immune system to respond more strongly to the TST after MTB infection. In this situation accepting the QFT-G response as the gold standard validates a 10 mm TST cut off for BCG vaccinated persons as the minimum induration size on which a decision for treatment for LTBI can be based.As one might expect, our study demonstrates – with a κ value of 0.08 – a poor correlation between the results of TST and those of the QFT-G assay among BCG-vaccinated contacts. However, it was surprising that even in unvaccinated contacts QFT-G failed to confirm nearly 50% of positive TST results – there were only 14 QFT-G-positives (52%) among 27 TST-positive contacts – when the induration cut off for the TST was set to 5 mm diameter. This indicates at least four possible explanations.The first is that QFT-G is not sensitive enough to confirm true LTBI, thus producing false negative IFN-γ -assay results. However, there is no evidence for this assumption, because in three contacts there were positive QFT-G results while their TST response was negative, whereas the rest of the TST/QFT-G-negative group showed excellent concordance 122/125, 97.6%). The second possibility is that the 5 mm cut off of TST representing a positive result, used to achieve increased sensitivity in high-risk persons, is too low, because of an overestimation of the true intensity of exposure of a contact and must therefore be raised. The fact that increasing the cut off to 10 mm resulted in a strong agreement (κ = 0.68) between the results from TST and those from QFT-G in the non-vaccinated population seems to support the influence of BCG as a confounder of the TST. There were only 3 (2%) QFT-G negative but TST positive responses >10 mm in the non-vaccinated group, and 6 (4%) QFT-G positive but TST negative, indicating QFT-G is equally or more sensitive than the TST in non-vaccinated persons. This corresponds to the results of other studies, in which a strong agreement between TST and ELISPOT could be seen when a diameter of 10 mm was used as cut off from the start [, 97.6%. M. avium or M. intracellulare) have been used in a large number of studies and countries to determine the rate of immune reactivity to NTM in various populations [The third possibility is that, as in the case of BCG vaccination, there are cross-reactivities with other mycobacteria. Antigen preparations from non-tuberculous mycobacteria  of the 125 TST-negative close contacts in our study population.Of course, some contacts are definitely known While sensitivity cannot be formally tested in LTBI, the comparison of QFT-G with TST in BCG unvaccinated contacts indicates similar sensitivity for LTBI. Limited studies  have shoIn conclusion, the data presented here suggest that a MTB-specific whole-blood INF-γ test (QFT-G) appears to be more valid method for screening recent contacts for LTBI, especially when a large number of contacts have previously been BCG-vaccinated or their BCG vaccination status cannot be accurately determined. QFT-G also has benefits over the TST if contacts have migrated from foreign countries, where NTM infections are prevalent. Owing to the high specificity of this IFN-γ test, the ESAT6/CFP-10/TB7.7 based QFT-G assay allows better discrimination between true infection and cross-reactivity, and can thus circumvent the unpredictable influence of BCG and NTM on the TST. Availability of MTB-specific whole-blood INF-γ tests – more accurate than the TST – could lead to a better chance of true positive test results and thus, in turn, to more systematic use of treatment for LTBI. In addition, it is suggested that if the TST is to be used, the cut off should be raised from 5 mm to 10 mm, even for close contacts, in order to minimize the large number of false positive results seen at this cut off.The author(s) declare that they have no competing interests.RD and TS designed the study and wrote the manuscript. RD and AN carried out and interpreted the statistical analysis. CL and MF participated in the design of the study and the data interpretation. KM recruited patients, obtained medical data and assessed results. All authors read and approved the final manuscript.